Published online before print December 8, 2008, doi: 10.1161/HYPERTENSIONAHA.108.121863
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(Hypertension. 2009;53:196.)
© 2009 American Heart Association, Inc.
Original Articles |
Functional Characterization and Transcriptome Analysis of Embryonic Stem Cell–Derived Contractile Smooth Muscle Cells
From the Center of Physiology and Pathophysiology (S.P.P., H.L., K.P., J.W., S.C., M.X.D., K.O., N.K., J.H., A.S.), Institute of Neurophysiology and Center of Molecular Medicine, University of Cologne, Cologne, Germany; and Max-Delbrueck-Center for Molecular Medicine (H.S., N.H.), Berlin, Germany.
Correspondence to Agapios Sachinidis, University of Cologne, Center of Physiology and Pathophysiology, Institute of Neurophysiology, Robert Koch Str 39, Cologne, Germany. E-mail a.sachinidis{at}uni-koeln.de
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Abstract |
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Complete transcriptome profiling of contractile smooth muscle cells (SMCs) differentiated from embryonic stem cells is crucial for the characterization of smooth muscle gene expression signatures and will contribute to defining biological and physiological processes in these cells. We have generated a transgenic embryonic stem cell line expressing both the puromycin acetyl transferase and enhanced green fluorescent protein cassettes under the control of the Acta2 promoter. Applying a specific monolayer culture protocol using retinoic acid, a puromycin-resistant and enhanced green fluorescent protein–positive Acta2+ SMC population of 95% purity was isolated. Acta2+ SMCs were characterized by semiquantitative and quantitative RT-PCR profiling of SMC markers and by microarray expression profiling, as well as by immunostaining for SMC-specific cytoskeletal proteins. Patch-clamp electrophysiological characterization of these cells identified SMC-specific channels such as the ATP-sensitive potassium channel and the Ca2+-activated potassium channel. Culturing of Acta2+ SMCs in serum-containing medium resulted in a significant number of hypertrophic and binucleated cells failing to complete cell division. Functional characterization of the cells has been proved by stimulation of the cells with vasoactive agents, such as angiotensin II and endothelin. We concluded that our embryonic stem cell–derived SMC population possesses the contractile and hypertrophic phenotype of SMCs incapable of proliferation. This is the first study describing the complete transcriptome of ES-derived SMCs allowing identification of specific biological and physiological processes in the contractile phenotype SMCs and will contribute to the understanding of these processes in early SMCs derived from embryonic stem cells.
Key Words: transcriptome • smooth muscle • embryonic stem cells • hypertrophy • contractile phenotype
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Introduction |
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Smooth muscle cells (SMCs) play an important role in the pathogenesis of cardiovascular diseases, such as atherosclerosis and hypertension.1 Under pathophysiological conditions, SMCs are able to undergo several phenotypic alterations in morphology, gene expression, contractile capacity, or proliferative capacity.1–3 The definition and identity of an SMC phenotype as nondividing "contractile" or as proliferative "synthetic" poses a major challenge because of the plasticity of SMCs, the heterogeneity of their phenotypes, and the underlying gene expression patterns modulating the SMC phenotype. Because of the phenotypic modulation during cell culturing from the contractile to synthetic one, the usefulness of the available primary SMC lines established from adult tissues for cardiovascular research under physiological conditions and for drug discovery is limited. Therefore, establishing a contractile SMC line is essential. We hypothesize that embryonic stem cell (ESC)–derived SMCs better reflect the contractile phenotype, because SMCs are developed from undifferentiated ESCs. Identification of the whole transcriptome using microarrays combined with in silico technologies offers a powerful systems biology approach to address a multitude of biological and physiological processes characteristic of different cell phenotypes.4,5
The origin and localization of SMCs in the cardiovascular system and the molecular mechanisms of SMC development are not well understood during in vivo development. ESCs offer an attractive model for studying differentiation and developmental processes toward generating very early SMCs.6
Retinoids are able to regulate differentiation of SMCs in vitro and are known to distinctly influence the phenotype of cultured SMCs.7,8 Retinoic acid (RA) and dibutyryl cAMP can induce embryoid bodies derived from mouse ESCs to differentiate into SMCs.9 For human ESCs, a highly efficient RA-based monolayer protocol for differentiation into SMCs has been reported.10 However, because of differences in the protocols and species used, different SMC phenotypes were observed. More recently, using the lineage selection approach, contractile Acta2- and Myh11-expressing cells have been isolated using the classic embryoid body model.3
We generated an aortic 
-2 smooth muscle actin (Acta2) ESC line allowing monitoring and quantification of SMCs by enhanced green fluorescent protein (EGFP), expression as well as isolation of Acta2+ SMCs by puromycin selection. ESC-derived Acta2+ SMCs were then functionally characterized and their transcriptomic identity was determined.
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Materials and Methods |
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Materials
Details on reagents, antibodies, plasmid construction, drugs, and the used methods are provided in the expanded Materials and Methods section in the online data supplement (available at http://hyper.ahajournals.org).
Culture of ESCs and Isolation of SMCs
CGR8 murine ESCs (ECACC 95011018) were cultured without feeder cells, as described previously.11
Construction of Reporter Plasmid With the Acta2 Promoter and EGFP and Generation of the Acta2 ESC Line
The 5.5-kb promoter region upstream of the translation initiation site of the Acta2 gene12 was isolated from BAC clone RP24–329P13 (BACPAC Resources Center) and subcloned into the ESC reporter construct pPuroIRES2-EGFP.
Electrophysiological Recordings
Membrane potentials were recorded in the whole-cell and cell-attached configurations of the patch-clamp technique using an EPC-9 amplifier (Heka Elektronik).
Affymetrix Experiments and Functional Annotation
Total RNA preparation quality was assessed by agarose-formaldehyde gel electrophoresis. Three independent total RNA preparations, each 1 µg from the Acta2+ SMCs, murine aortic SMCs, 15-day-old differentiated ESCs, and undifferentiated ESCs, were labeled with the 1-cycle target labeling and control reagent package (Affymetrix), as described in the manufacturer’s instructions. After fragmentation of the cRNA using the standard Affymetrix protocol, 15 µg fragmented complementary RNA was hybridized for 16 hours at 45°C to Mouse Genome 430 2.0 arrays carrying 45 101 probe sets (for statistical and GO analysis of the data, see the data supplement).
Immunocytochemistry
On day 15, the enriched population of SMCs was washed with PBS and trypsinized, and a single-cell suspension was plated onto 0.2% gelatin-coated cover slips using differentiation medium containing 2.5 µg/mL of puromycin. The immunostaining of SMC-specific proteins is described in the data supplement.
Semiquantitative RT-PCR Analysis, Quantitative Real-Time PCR, Flow Cytometry, and Contractile Experiments
Methods are described in the data supplement.
Determination of Cell Number and Cell Volume of Acta2+ SMCs
Day 15 Acta2+ SMCs and control murine aortic SMCs were seeded in 6-well culture dishes and cultured in differentiation medium. After 3 and 5 days of cultivation, cells were trypsinized, and cell number and cell volume were determined using the CASY-1 cell analyser (Schärfe), as described previously.13
Contractility Experiments
Acta2+ SMCs were stimulated with angiotensin II (Ang II), endothelin 1, and 50 mmol/L of KCl as described in the data supplement.
Statistical Analysis
To evaluate statistical significance of electrophysiological recordings and the proliferative capacity of Acta2+ SMCs, Student t test was performed where applicable. P<0.05 was considered significant. Results are given as means±SEMs or as SDs, respectively.
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Results |
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Isolation of Acta2+ SMCs From the Transgenic ESCs
A transgenic Acta2 ESC lineage was generated by stable transfection with the linearized pActa2p-Puro IRES2 EGFP construct. For induction of differentiation, the undifferentiated Acta2 ESCs (see Figure 1A and 1B, day 0, –RA) were cultured in the absence (see Figure 1A and 1B, day 15, –RA) and presence of all-trans-RA (see Figure 1A and 1B, day 15, +RA) or in the presence of puromycin after 10 days of RA treatment (see Figure 1A and 1B, day 15, +RA+pur) for an additional 5 days. Activity of the Acta2 promoter as detected by EGFP fluorescence increased significantly after 6 days. As demonstrated in Figure 1B, RA induced differentiation of Acta2 ESCs to Acta2-expressing SMCs, but in the absence of puromycin, cell colonies with the typical cell morphology of undifferentiated cells remained (Figure 1B, middle, arrow). In contrast, after day 10 of differentiation, when cells were treated with puromycin for an additional 5 days, only Acta2- and EGFP-expressing SMCs were observed. These SMCs (see Figure 1B, day 15, +RA+pur) showed a more spindle-like shape when compared with murine aortic SMCs (see Figure 1B, aortic SMCs). The amount of Acta2-expressing SMCs in the 15-day-old differentiated Acta2 ESCs in the absence of RA (15-day-old Acta2 ESCs), in the presence of RA, or in the presence of both RA (for 10 days) and puromycin (for 5 days) as quantified by fluorescence-activated cell sorter analysis was 0.2%, 83.0%, and 95.0%, respectively (Figure 1C). The purified Acta2-expressing cells are called, hereafter, Acta2+ SMCs.
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Characterization of the Acta2+ SMCs by RT-PCR Detection of Smooth Muscle–Specific Genes and Immunostaining of Smooth Muscle–Specific Cytoskeletal Proteins
RT-PCR analysis indicated a maximal expression level of Acta2 (also known as -smooth muscle actin), Tagln (also known as SM22), and Myh11 (also known as smooth muscle myosin heavy chain) compared with the undifferentiated Acta2 ESCs and with 15-day-old differentiated Acta2 ESCs (Figure 2A; for RT-PCR conditions and primers, please see Table S1). Acta2 and Tagln are accepted as canonical markers for early SMCs, whereas Myh11 serves as a canonical marker for late SMCs. Aortic SMCs also expressed the smooth muscle–specific genes but the expression level, especially of Myh11, was markedly lower than in the Acta2+ SMCs. Not surprisingly, Zfp42 (also known as REX1) and Pou5f1 (also known as Oct4), 2 canonical markers for pluripotent cells, were not expressed in either SMC population but were highly expressed in undifferentiated and in 15-day-old differentiated ESCs. Smooth muscle–specific cytoskeletal proteins, such as calponin, Myh11, Acta2, and transgelin, were detected by immunocytochemistry as well (Figure 2B).
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Electrophysiological Recordings
To functionally characterize Acta2+ SMCs, their typical membrane potential was measured (Figure 3). With the whole-cell configuration, the membrane potential recorded was –38.01±1.22 mV (n=20). To characterize the hormonal regulation, SMC vasoconstrictors carbachol, epinephrine, and Ang II14,15 were applied to single patch-clamped cells (Figure 3A). All 3 of the agonists depolarized the membrane potential significantly (Figure 3A). It is well established that the vasoconstriction induced by these agonists is mediated by the elevation of intracellular free Ca2+ (for review see References 14 and 15). Increase of the intracellular free Ca2+ in contractile SMCs occurs mainly via voltage-dependent Ca2+ channels and, to a lesser extent, via nonselective cation channels at the plasmalemmal membrane or via internal Ca2+ stores, such as the ryanodine and the inositol triphosphate receptors found in the sarcoplasmic reticulum membranes.14,15 Activity of different ion channels present at the plasma membrane, such as the K+, Cl–, and cation channels, controls the membrane potential, thereby affecting the voltage-dependent Ca2+ channel activity and calcium entry.14,15 In this context, opening of the smooth muscle–specific ATP-sensitive potassium channels (also known as IKATP channels) and the large-conductance Ca2+-activated K+ channels hyperpolarizes the membrane and promotes closure of voltage-dependent Ca2+ channels and, thus, opposes vasoconstriction.14,15 Recently, it has been shown that Ang II and epinephrine,14,15 as well as carbachol,16,17 inhibit the smooth muscle–specific IKATP channels and the large-conductance Ca2+-activated K+ channels, thereby inducing depolarization and contraction of the SMCs.
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We further characterized the presence of specific ion channels in ESC-derived Acta2+ SMCs. The presence of IKATP and IKca channels in the Acta2+ SMCs was detected after activation of the cells with chromakalim, an activator (ie, opener) of the ATP-sensitive K+ channels in several cell types, including SMCs,18 and with thapsigargin, a selective inhibitor of sarcoendoplasmic reticulum Ca2+-ATPase.19 As shown in Figure 3B, chromakalim (10 µmol/L) hyperpolarized the membrane potential from –42.78±5.14 mV to –47.74±5.01 mV (n=6). This effect was inhibited by subsequent application of glibenclamide (10 µmol/L), a potent IKATP-specific blocker.20 Thapsigargin (10 µmol/L) hyperpolarized the membrane potential from –33.26±4.11 mV to –46.68±6.77 mV (n=5), which was abolished by 1 µmol/L of iberiotoxin, a potent specific IKca blocker,21 demonstrating the presence of IKca. The activation of IKATP and IKca, hyperpolarization of the membrane potential, and reversal of this effect with channel-specific blockers clearly indicate that the Acta2+ SMCs possess channels that are specific for SMCs of the contractile phenotype.
Agonist-Induced Contraction of Acta2+ ESCs
To examine the contractile properties of Acta2+ ESCs, cells were stimulated with Ang II (1 µmol/L), endothelin (10 nmol/L), or KCl (50 mmol/L). Video files 1, 2, and 3 provide the time-lapse videos for a period of 15 minutes showing the cells after stimulation with Ang II, endothelin, and KCl, respectively (please see the data supplement). As indicated, stimulation of the cells resulted in contraction of the Acta2+ SMCs.
Microarray Expression Profiling and Validation of the Microarray Data
RNA from undifferentiated ESCs, 15-day-old differentiated Acta2 ESCs, from Acta2+ SMCs, and from murine aortic SMCs was used for hybridizations to Affymetrix MG 430 2.0 microarrays. Affymetrix expression data were verified by quantitative real-time PCR. As indicated in Figure S1 (please see the online data supplement), results from the Affymetrix analyses correspond well with the results obtained with quantitative real-time PCR.
Gene Expression Pattern in Acta2+ SMCs, Aortic SMCs, Undifferentiated ESCs, and 15-Day Differentiated ESCs
After RMA normalization of the 45 101 probe sets and removing nonexpressed or low-expressed transcripts, the remaining 27 435 transcripts were analyzed using principal component analysis of the 4 different transcriptomes (undifferentiated Acta2 ESCs, 15-day-old differentiated ESCs, Acta2+ SMCs, and aortic SMCs) to classify the variance in the data set. The 4 transcriptomes were well separated with experimental replicates showing little differences. In principal component (PC) 1 (accounting for 42.2% of variance in our data set), both aortic and Acta2+ SMCs grouped together closely, indicating a higher degree of similarity to each other than to undifferentiated Acta2 ESCs and the 15-day-old control population (Figure 4A). In PC 2 (accounting for only 28.8% of variability), the relation between aortic and Acta2+ SMCs was more distant (Figure 4A). ANOVA analysis (false discovery rate: 0.1%) identified 21 826 differentially expressed transcripts. K-Mean clustering of the differentially expressed probe sets resulted in 12 different expression profiles, as demonstrated by the heat map (Figure 4B). In this context, of particular importance is the characterization of common gene expression signatures in the Acta2+ and the aortic SMCs represented in clusters 2 and 5 (upregulated) and cluster 10 (downregulated) and the characterization of gene signatures specifically regulated in the Acta2+ SMCs in cluster 8 (upregulated) and cluster 11 (downregulated).
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To identify Acta2+ SMC-specific signatures in more detail, transcripts of the clusters above were further analyzed using the DAVID bioinformatics resource to identify enriched functional annotation terms in the categories Gene Ontology (GO) "Biological Process" (GOTERM_BP), "Molecular Function" (GOTERM_MF), and "Cellular Component" (GOTERM_CC; level 5), as well as the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Biocarta pathways.
Upregulated Transcripts in Acta2+ and Aortic SMCs
GOterm enrichment analysis with transcripts of cluster 2 and 5, which represent transcripts with a high expression level in Acta2+ and aortic SMCs, is shown in Table S2 (top). Several muscle and cytoskeleton specific GOs and signal transduction pathways have been identified. GOterm enrichment analysis of the cluster 8 transcripts with a higher expression level exclusively in Acta2+ SMCs is shown in Table S2 (bottom). Several muscle and cytoskeleton GOs and signal transduction pathways have been identified.
Downregulated Transcripts in Acta2+ and Aortic SMCs
Table S3 (top) shows the most predominant GO categories and pathways that are enriched among the probe sets, with a lower expression level in both the Acta2+ and aortic SMC populations compared with the 15-day-old differentiated and undifferentiated Acta2 ESCs (cluster 10). As shown, these GOs and pathways are associated with the process of transcription (zinc ion binding, positive regulation of transcription) and cell proliferation (M phase of the mitotic cell cycle). GO term enrichment analysis with the transcripts of cluster 11 showing a low expression level exclusively in Acta2+ SMCs is shown in Table S3 (bottom). GOs such as M phase and pathways such as the cell cycle, which are associated with a high proliferation rate, were markedly enriched.
Proliferative Capacity of Acta2+ SMCs
Several genes participating in the progression of cell cycle were downregulated in the Acta2+ SMCs, suggesting a low proliferative capacity. We, therefore, studied the actual proliferative capacity of these cells. In addition, based on the GOs associated with an increase of cell size via hypertrophy, we expected that, after prolonged culture of Acta2+ SMCs, cells should become larger because of hypertrophy. As clearly demonstrated in Figure 5A, the number of cells in the S phase indicated by Ki67 staining is very limited. Few cells were positive for Ki67 after 24 hours of culture. After 3 days and 5 days of cultivation, a reduction of the cell number to approximately half of the initial number was observed (Figure 5B). At the same time, the cell volume of Acta2+ SMCs increased by 37% after 3 or 5 days of cultivation (Figure 5C). In contrast, aortic SMCs were hyperplasic and proliferated >4-fold after 3 days of culturing concomitant with a slightly reduced cell volume. In the Acta2+ SMC population, binucleated cells were observed after 3 and 6 days of subculturing (Figure 5D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our findings clearly indicate that the Acta2+ SMCs express classical cytoskeletal smooth muscle genes and proteins emphasizing the reliability of the gene signatures of the ESC-derived SMCs. We characterized the electrophysiological properties of Acta2+ SMCs by detecting IKATP and IKca hyperpolarization of the membrane potential and reversal of this effect with channel-specific blockers. These results suggest that the Acta2+ SMCs possess channels that are specific for SMCs of the contractile phenotype. Similar to SMCs derived from D3 ESCs, the ESC-derived Acta2+ SMCs are functional, responding to various contraction-inducing agonists.3
As expected, the observed predominant upregulated gene expression signatures in the Acta2+ and the aortic SMCs compared with the undifferentiated and the 15-day-old differentiated ESCs involve classic SMC cytoskeletal, blood vessel, and muscle developmental genes. Notably, we identified "apoptosis," "small GTPase-mediated signal transduction," "negative regulation of cell proliferation," and "regulation of cell growth," as well as several signal transduction pathways (including the transforming growth factor β1), as commonly upregulated gene signatures in both SMC populations (please see Table S2). Apoptosis is a crucial process counteracting the cell division in intact normal blood vessels and is essential for regulating vascular remodeling during cardiovascular diseases (for review, see Reference 22). Recently, it has been shown that Notch signaling represses SMC differentiation from the contractile to the proliferative phenotype and maintenance of the contractile SMC phenotype.23 Small monomeric G proteins, such as Ras, control cell proliferation, differentiation, and apoptosis via activation of Raf proteins and mitogen-activated protein kinases.24 These findings suggest a key role of the transforming growth factor β signaling transduction pathway for the SMC type. Transforming growth factor β is a potent vascular smooth muscle differentiating factor and has been demonstrated to elevate the expression of Acta2, smooth muscle myosin, and calponin.25 Furthermore, the "negative regulation of cell proliferation," and the "regulation of cell growth in size" signatures clearly correlate with the lower proliferative capacity of the SMCs, showing a hypertrophic phenotype as compared with undifferentiated and 15-day-old differentiated cells. Gene expression signatures that are exclusively upregulated in the Acta2+ SMCs are related to the actin cytoskeleton, smooth muscle contraction, and vasculature development. Interestingly, we observed specific gene signatures related to the ErbB, Wnt, epidermal growth factor, and the Ang II signal transduction pathways that are involved in proliferation and hypertrophy of SMCs. "Nuclear factor of activated T cells and hypertrophy of the heart" (10 genes, including endothelin 1 and cardiotrophin) was a unique gene signature for the Acta2+ SMCs, suggesting a hypertrophic SMC phenotype. Hypertrophy is recognized as a characteristic phenomenon in cardiomyocytes and SMCs of the contractile phenotype and occurs because of increased protein synthesis without cell division.1,2 In this context, it has been shown that endothelin 1 promotes vascular SMC hypertrophy.26 The nuclear factor of activated T-cell signaling controls multiple steps in the development of the cardiovascular system, including peripheral vascular development during angiogenesis (for review, see Reference 27).
Fifteen genes belonging to the Wnt signal transduction pathway are exclusively upregulated in the Acta2+ SMCs.28 Recently, it has been shown in the mouse model that the Wnt/β-catenin pathway is required for coronary vessel formation.29 Surprisingly, we found 36 genes and 6 genes belonging to the GO categories "neurogenesis" and "neural crest cell differentiation," respectively. These findings suggest that the Acta2+ SMCs might be derived from precursors that are capable of both neuronal and SMC differentiation. In this context, it has been shown recently that neural crest cells are able to differentiate into cell types expressing cytoskeletal markers characteristic of SMCs.30,31
Several genes related to cell proliferation were exclusively downregulated in the Acta2+ SMCs (see Table S3). Therefore, we expected that Acta2+ SMCs are incapable of proliferating but competent to grow because of hypertrophy. Our proliferation data clearly demonstrate that, in contrast to aortic SMCs, the Acta2+ SMCs are incapable of proliferation and cytokinesis. The phenomenon of the acytokinesis is well known during embryonic heart growth. During embryogenesis, heart mass is mainly increased via hyperplasia of the cardiomyocytes.32 After birth, the heart mass increases because of a transition from hyperplasia to hypertrophy, a process marked by binucleation, where cardiomyocytes lose their ability to complete cytokinesis.33
Perspectives
Applying a lineage selection approach, we isolated purified functional Acta2+ SMCs possessing the native contractile phenotype and identified SMC-specific gene expression signatures, including signal transduction pathways. As opposed to the available, nonphysiological synthetic proliferative phenotype of SMCs, the ESC-derived contractile Acta2+ SMCs are ideal for in vitro studies of biological, physiological, cellular, and molecular processes. This cell line allows for reproducible generation of an unlimited number of contractile SMCs that are required for consistent and reliable experimental research into basic intracellular signal transduction mechanisms involved in the development of hypertension and other vascular diseases. In addition, these cells offer an optimal model for drug discovery for treatment of vascular diseases. The identification of gene signatures and signal transduction pathways that are specifically expressed in the Acta2+ SMCs will contribute to a gene expression atlas for SMCs and will enable functional studies to elucidate their role in SMC differentiation processes.
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Acknowledgments |
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We thank Dr Gary K. Owens, Department of Molecular Physiology and Bological Physics, University of Virginia School of Medicine (Charlottesville, Va), for providing the primary mouse vascular SMCs.
Source of Funding
This work was supported by a grant from the European Community (Sixth Framework Programme, Thematic Priority: Life Sciences, Genomics and Biotechnology for Health, contract FunGenES LSHG-CT-2003-503494) to the Institute of Neurophysiology and the Max-Delbrueck-Center.
Disclosures
None.
Received August 20, 2008; first decision September 13, 2008; accepted November 11, 2008.
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